Production of conjugates

ABSTRACT

A method of reacting a first chemical entity and a second chemical entity to form a conjugate in which the first and second chemical entities are covalently bound with respect to each other, comprises bringing into simultaneous contact the first chemical entity, the second chemical entity and a thiol generator, wherein the thiol generator reacts with the first chemical entity in a thiolation reaction resulting in formation of a free sulfhydryl group on the first chemical entity, and the free sulfhydryl group reacts with the second chemical entity to form the conjugate, and wherein the second chemical entity is polyvalent with respect to its reactivity with sulfhydryl groups. The present invention primarily differs from the prior art in that no separation step is involved between reaction of the thiol generator and first chemical entity and reaction with the second chemical entity. The invention also provides a conjugation kit.

RELATED APPLICATIONS

This application is a U.S. National Phase Application of InternationalApplication No. PCT/GB2006/004633, filed Dec. 12, 2006, which claims thebenefit of GB Applications 0525223.4, filed Dec. 12, 2005, and0614533.8, filed Jul. 21, 2006, all of which are incorporated herein intheir entirety.

FIELD OF THE INVENTION

This invention relates to the production of conjugates and concerns amethod and a kit for performing the method.

BACKGROUND TO THE INVENTION

Conjugates are widely used in bioscience research, diagnostics andmedicine. In the simplest case conjugates take the form of a firstchemical entity (A), typically a molecule such as a biomolecule, that islinked to a second chemical entity (B), such as a label molecule, toform an AB hybrid. Oligomeric forms, represented by the formulaA_(j)B_(k), where j and k are integers, are also possible. Conjugatesare usually designed for a specific purpose and often involve novelcombinations of materials that are not naturally occurring. Typically,one component of the conjugate has the capacity to interact with othermolecules (e.g. antigens), e.g. being an antibody, and the secondcomponent adds some other useful property (e.g. measurability, abilityto kill cancer cells), e.g. being a label.

Conjugates of the present invention may comprise combinations ofentities, where A and/or B may comprise one of the following:antibodies, antibody fragments, nucleic acids, beads, polymers,liposomes, carbohydrates, fluorescent proteins and dyes, peptides,radionuclides, toxins, gold particles, streptavidin, biotin, enzymes,chelating agents, haptens, drugs and many other molecules. This listencompasses a vast array of molecules and thus the number of possiblecombinations in conjugates is almost limitless. It follows that there isconsiderable scope to create novel hybrid molecules with unusual orunique properties.

One of the most important applications of immunoconjugates is in thequantitation and/or detection of antigens, which are often presented ona surface. For example, in western blotting applications the antigen isimmobilised on a sheet of nitrocellulose; in an enzyme-linkedimmunadsorbent assay (ELISA), the antigen is adsorbed on surface of apolystyrene plate; in immunohistochemistry, the antigen is embedded,along with many other proteins, in a thin slice of tissue, which isattached to a glass slide. While these techniques differ fundamentallyin the way in which the antigen is presented to the conjugate, thechoice of detection methods is essentially the same. There are two maintypes. With direct detection, the ‘primary’ antibody (i.e. the antibodythat binds to the antigen) is conjugated to a label that can be measuredwith a suitable measuring device. With indirect detection, the label isintroduced via a secondary reagent, which binds to the primary antibody.The secondary reagent most often is an antibody conjugate comprising asecondary antibody conjugated to a label. More complex detectionstrategies exist but each of these generally is a variation on one ofthe above two themes.

With indirect methods, one secondary reagent can be used with a range ofunlabelled primary antibodies, which is extremely convenient, althoughthe need for more incubation and wash steps than with direct methods isa major disadvantage. There is also potential for unwantedcross-reactivity of the secondary antibody with immobilised antigens.While direct detection methods offer considerable advantages in terms ofspeed, cost, and data quality, indirect methods currently predominate.The explanation for this fact is that most primary antibodies areavailable commercially only in an unlabeled form. Moreover, thesereagents are expensive and usually cannot be purchased by researchers inquantities that allow cost-effective production of labelled conjugatesusing current labelling methodologies.

In order to produce a conjugate, a bifunctional reagent that containstwo reactive groups is generally used to link the two components ofinterest. The reactive groups on the bifunctional reagent are eitheridentical in functionality (‘homobifunctional’) or different infunctionality (‘heterobifunctional’). The best-known example of ahomobifunctional reagent is the bis-aldehyde glutaraldehyde, whichreacts with amines (or hydrazides). Since most biomolecules containmultiple amines, the use of glutaraldehyde commonly results in theformation of high molecular weight conjugates. Furthermore, thepolymeric nature of solutions of glutaraldehyde, which can varyconsiderably with age, means that conjugates prepared withglutaraldehyde are generally quite difficult to reproduce.

Heterobifunctional reagents are generally preferred in the preparationof conjugates as they allow the operator to exert a higher degree ofcontrol over the conjugation process. A popular heterobifunctionalconjugation strategy involves the coupling of an amine group on onemolecule (B) to a free sulfhydryl group (SH) on another molecule (A) viaa heterobifunctional reagent (X-Y) having an amine-reactive moiety (X)and a sulfhydryl-reactive moiety (Y). A ‘spacer’ often separates thereactive moieties of the heterobifunctional reagent; there are manyheterobifunctional reagents that have varying spacer structures butwhich share essentially the same chemical reactivity.

Typically, one biomolecule (B) to be conjugated is reacted via its aminegroups with the X functionality of the heterobifunctional reagent,resulting in a B-Y derivative. Excess heterobifunctional reagent is thenremoved and purified B-Y is reacted with sulfhydryl groups on the othermolecule (A). X is commonly an N-hydroxysuccinimide (NHS) ester, while Ymay be one of several moieties. Y may or may not be integrated into thefinal AB conjugate. The sulfhydryl group derived from A is almost alwaysincorporated either as a stable thioether bond or as one half of areversible (reducible) disulphide bridge between A and B. Y may be anysulfhydryl-reactive functionality including: maleimide, epoxide,iodoacetyl, bromoacetyl, pyridyldithiol, methanethiosulfonate, and thelike.

Examples of amine and sulfhydryl reactive heterobifunctional reagentsinclude: N-succinimidyl 3-(2 pyridyldithio) propionate (SPDP); variantsof SPDP with extended spacers (LC-SPDP; LC=‘long chain’) and sulfogroups to increase aqueous solubility (sulfo-LC-SPDP);succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT);sulfo-LC-SMPT;Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC);sulfo-SMCC; m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);sulfo-MBS; N-Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB); sulfo-SIAB;Succinimidyl-4-(p-maleimidophenyl)butyrate (SMBP); sulfo-SMBP;N-(γ-Maleimidobutyryloxy)succinimide ester (GMBS); sulfo-GMBS;Succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX); and its extendedspacer form SIAXX; Succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (SIAC); and itsextended spacer form (SIACX); p-Nitrophenyl iodoacetate (NPIA). Thereare many other related examples, such as the carbonyl andsulfhydryl-reactive linker, β-maleimidopropionic acid hydrazide (BMPH).

The sulfhydryl groups on A may be indigenous. However, more commonlysulfhydryl groups are not present and need to be introduced by athiolation reaction prior to the conjugation step. In the case ofantibodies, thiol groups may be generated by means of a reducing agent(e.g. MEA or dithiothreitol (DTT)), which break disulfide bridges atvarious positions on the antibody molecule. Alternatively, techniquesare known by which other functional groups (commonly amines) can bemodified to introduce either a free sulfhydryl group or a protectedsulfhydryl group, which can then be deprotected by treatment with areducing agent to generate a thiolated product (i.e. A-SH). In the knownconventional techniques, prior to conjugation with B-Y at least oneseparation step is required to separate the desired thiolated productA-SH from unreacted thiolation reagent, and any by-products includingfree sulfhydryl groups that would compete for conjugation to B-Y andpossibly also reducing agent that would otherwise compete with A-SH forconjugation to B-Y. Separation is performed by techniques includingdesalting on chromatography columns, gel filtration, dialysis, orwashing. The separation step or steps inevitably result in losses anddilution of material. Because of the tedious nature of the separatingstep(s) and/or requirement for significant quantities of A, thethiolation step may never be thoroughly optimised.

By way of example, 2-iminothiolane (2IT), which is also known as Traut'sreagent (Traut et al., Biochemistry 12, 3266-3273, 1973) has previouslybeen used to introduce SH groups into proteins, particularly antibodies.The reagent reacts with primary amines (e.g. present on lysine) andgenerates a terminal sulfhydryl group in a ring-opening reaction. ExcessTraut's reagent is removed, typically by desalting, prior to conjugationof the resulting thiolated molecule with a thiol-reactive group onanother molecule. Although not mentioned in otherwise comprehensiveworks on bioconjugation chemistry (e.g. Bioconjugate Techniques; G. THermanson, Academic Press 1996), 2IT also undergoes a secondary reactionin which the nascent thiol reacts intramolecularly to form an unreactivethioester (Bartlett & Busch., Biol. Mass Spectrom. 23, 353-356, 1994;Singh et al. Anal. Biochem. 236, 114-125, 1996). It is clear from theknown chemistry of Traut's reagent that the duration of the thiolationreaction may be critical to the success of the subsequent conjugationstep, and that desalting or other separation steps need to be completedquickly.

Conventional prior uses of 2IT in the production of conjugates withmolecules engineered to contain thiol-reactive functions employ excess2IT followed by a desalting, dialysis or wash step. This type ofapproach is recommended by suppliers of 2IT (e.g. Pierce technicalbulletin 0414; product 26101) and of products used in the preparation ofbioconjugates (e.g. Prozyme TechNote #TNPJ300). Other publications thatdescribe this approach include: U.S. Pat. Nos. 6,962,703, 6,936,701,6,669,938, 6,010,902, 5,869,045, 5,164,311; Stanisic et al., Infectionand Immunity 71, 5700-5713, 2003; Mandler et al., Journal of theNational Cancer Institute, 92, 1573-1581, 2000; Huwyler et al., ProcNatl Acad Sci 93, 14164-14169, 1996.

One potentially promising solution to the problem of desalting wassuggested (Haughland. Handbook of Fluorescent Probes and ResearchChemicals, 6^(th) edition, Molecular Probes, p 49) which involvedreduction of protected sulfhydryl groups by TCEP(Tris(2-carboxyethyl)phosphine). While it is claimed that removal ofTCEP is unnecessary, as it does not interfere with subsequentconjugation steps, Getz et al (Anal Biochem 273, 73-80, 1999) showedsignificant interference of TCEP in conjugation reactions. Moreover,Shafer et al (Anal Biochem 282, 161-164, 2000) reported that TCEPcombines rapidly with the sulfhydryl-reactive maleimide and iodoacetylgroups. Furthermore, bioconjugation reactions commonly are carried outin phosphate buffers at pH 7-8, under which conditions TCEP is unstable(Han & Han, Anal Biochem 220, 5-10, 1994). TCEP is very stable atextremes of pH (e.g. in 10 mM HCl or in 100 mM NaOH), which are notcompatible with most biomolecules. While TCEP has found certain nicheapplications its serious limitations have ensured that the preferredmethods for producing bioconjugates have changed little since it becamecommercially available in 1992. TCEP does not contain a sulphur atom andtherefore, for present purposes, is not considered a “thiol generator”.

McCall et al (1990 Bioconjugate Chem. 1, 222-226) disclosed a one stepmethod for conjugating macrocyclic chelators to antibodies using 2IT.Specifically they used 2IT to join6-[p(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N¹,N¹¹,N¹¹¹-tetraceticacid, abbreviated as BAT, or a similar compound,2-[p(bromoacetamido)benzyl]-1,4,7,10-tetraazacyclododecane-N,N¹N¹¹,N¹¹¹-tetraacetic acid (abbreviated as BAD), to a mouse antibody. The BAT/BADreagents were monovalent with respect to sulfhydryl reactive groups i.e.having only one group per molecule able to react with a sulfhydrylgroup. McCall et al suggested that the “one step” method disclosedtherein was applicable only to the particular BAT/BAD reagent (“sinceunder mildly alkaline conditions bromoacetamide reagents react rapidlywith sulfhydryl groups but only slowly with amino groups, the antibody,BAT and 2 IT solutions could be combined in a single reaction mixture”).There is no suggestion that this technique might be generally applicableand the standard method used commercially remains a 2 step approach withan intervening desalting, purification or washing stage.

SUMMARY OF THE INVENTION

In one aspect the present invention provides a method of reacting afirst chemical entity and a second chemical entity to form a conjugatein which the first and second chemical entities are covalently boundwith respect to each other, comprising bringing into simultaneouscontact the first chemical entity, the second chemical entity and athiol generator, wherein the thiol generator reacts with the firstchemical entity in a thiolation reaction resulting in formation of afree sulfhydryl group on the first chemical entity, and the freesulfhydryl group reacts with the second chemical entity to form theconjugate, and wherein the second chemical entity is polyvalent withrespect to its reactivity with sulfhydryl groups (i.e. one molecule ofthe second chemical entity can react with two or more sulfhydrylgroups).

The present invention differs from conventional prior art techniques inthat no separation step is involved between reaction of the thiolgenerator and first chemical entity and reaction with the secondchemical entity. Instead, in the present invention, the three materialsare in simultaneous contact at some stage in the conjugation procedure,with the thiol generator acting to produce free sulfhydryl groups on thefirst chemical entity while in contact with both entities to beconjugated. There is no separation or partial separation of thethiolated first chemical entity from excess thiol generator or from anyby-products that might be formed prior to contact with the secondchemical entity. An advantage of this approach is that the newly formed,though labile, SH groups can react immediately with the second chemicalentity.

The invention is based in part on the realisation that a separation stepis not necessary, and that the consistent use of separation in the priorart is based on a misconception. By eliminating the separation step(s)of prior conjugation procedures, the method of the invention issubstantially simplified. Further, because there is no separation stepwith inevitable loss of material, the conjugation reaction of theinvention can be performed using very small amounts of materials. Theinvention thus paves the way for easy formation of conjugates in theform of labelled reagents useful in direct assays, e.g. facilitatingdirect labelling of almost any protein, on any scale, and offers furtherbenefits of immunoassay simplification, greater reproducibility and costreduction.

The thiol generator (TG) contains one or more sulphur atoms and reactswith the first chemical entity (e.g. by ring opening, rearrangement orotherwise) to produce a covalently bound sulfhydryl (or thiol) group onthe first chemical entity, the sulfhydryl group including a sulphur atomfrom the thiol generator. The thiol generator conveniently comprises athiolactone (see below) and/or an iminothiolactone (see below) and/or anepisulfide such as 1,2-epithiopropane and/or a thiazolidine such as2-[(4-dimethylamino)phenyl]-1,3-thiazolidine and N-substituted analoguesof thiazolidines, where the said N-substituents may be introduced tomodify the ring-opening properties (Canie et al., Pure & Appl. Chem. 68,813-818, 1996). A mixture of materials may be used. Suitablethiolactones include N-acetylhomocysteinethiolactone (NAHCT) (Benesch &Benesch. Proc. Natl. Acad. Sci. 44, 848-853, 1958). Suitableiminothiolactones include 2-iminothiolane (2IT), also known as Traut'sreagent, which is commercially available as 2-iminothiolanehydrochloride. Substituted and derivatised materials may also be used,e.g. 5- and 4,5-alkyl substituted 2-iminothiolane (Goff and Carroll.Bioconjugate Chem. 1, 381-386, 1990). Possible variants include5-methyl-; 5-tert-butyl-; 5-phenyl-; 5,5-dimethyl-; 5-spiro-; and4,5-ring analogues.

The first chemical entity (A) includes a chemical functionality thatreacts with the thiol generator to produce a thiolated version of A,A-SH. The functionality is generally a nucleophilic group, typically anamine, particularly a primary amine, or a hydroxyl group. Typicalthiolation reactions are as follows:

2IT is fully water-soluble and reacts with primary amines in the pHrange 7 to 10. In conventional conjugate formation reactions, 2IT isused at a pH of about 8, under which conditions 2IT reacts efficientlyand rapidly with primary amines, e.g. in lysine residues present inpeptides, polypeptides and proteins. For reaction with primary amines,it has now been found that it is preferable to react 2IT at a pH lowerthan the conventional valve of 8. Thus when using 2IT the conjugationreaction is preferably carried out at a pH less than 8, preferably lessthan 7.8 and more preferably less than pH 7.7. A preferred pH range is7.0-7.5. Since the reactions of thiols with many types of thiol-reactivegroups Y take place efficiently between pH 6.5 and 7.5, it isundesirable to use TG at high pH values where competing hydrolysisreactions generate undesirable free thiols. Moreover, Y may also besubject to hydrolysis reactions at alkaline pH, as discussed below, ormay show reduced selectivity for thiols, as in the case of the popularmaleimide functional group. At higher pH, e.g. about 10, 2IT is alsoreactive with aliphatic and aromatic hydroxyl groups, although the rateof reaction with these groups is only about 0.01 that of primary amines(Alagon & King. Biochemistry 19, 4341-4345, 1980). In the absence ofamines, carbohydrates such as agarose or cellulose membranes can bemodified with 2IT to contain sulfhydryl residues. Polysaccharidesmodified in this manner are effective in covalently cross-linkingantibodies for use in immunoassay procedures.

The first chemical entity (generally referred to herein as “A”) istypically a polymer, preferably a biomolecular polymer (i.e. a polymericmolecule which occurs naturally in one or more living systems). Apreferred biomolecular polymer is a polypeptide. Desirably, but notessentially, the first chemical entity comprises or consists of anantibody or an antigen-binding fragment, such as Fab, Fv, scFv or asingle domain antibody, or a multimer of an antibody or antigen-bindingfragment thereof. Other examples of A include Streptavidin, neutravidin,protein A, polypeptide receptor molecules, and polypeptide ligands. Afirst chemical entity comprising one or more thiol groups may berepresented as A-SH.

Where the first chemical entity includes more than one chemicalfunctionality that reacts with TG, e.g. several primary amines, morethan one sulfhydryl group will be formed on the first chemical entity.

The second chemical entity (generally referred to herein as “B”)includes a plurality of sulfhydryl-reactive functional groups (Y) thatreact with the sulfhydryl group formed on the first chemical entity,resulting in production of the conjugate. The second chemical entity maythus typically be represented as B-Y. During the conjugation reactionthe sulphur atom of A-SH is usually integrated into the final conjugateas a stable thioether bond or one half of a reversible (reducible)disulphide bridge.

The second chemical entity includes more than one sulfhydryl-reactivefunctional group which may have identical or different chemistries.Sulfhydryl-reactive entities include maleimide, epoxide, iodoacetyl,bromoacetyl, pyridylolthiol, etc. The plurality of sulfhydryl-reactivefunctional groups (Y) may be naturally present in the second chemicalentity, but commonly it will be necessary for one or more of thesulfhydryl-reactive functional groups to be introduced to a molecule Bin a preliminary step to produce the second chemical entity. Suitableintroduction techniques are well known to those skilled in the art.

The second chemical entity (B-Y) typically comprises or includes a labele.g. enzyme, fluorescent material etc. for identification or measurementof materials via binding of the conjugated first chemical entity, or atoxin, therapeutic agent etc. for targeted delivery via binding of theconjugated first chemical entity.

The present invention provides a method which is generally applicable.Nevertheless, in preferred embodiments, the second chemical entity is orcomprises a polymer. In preferred embodiments the second chemical entitycomprises a polypeptide.

In a preferred embodiment the second chemical entity comprises anenzyme. Examples of preferred enzymes include HRP, alkaline phosphataseand glucose oxidase. HRP is especially preferred.

The functions of the first and second chemical entities may be reversed.

Since thiols that do not originate from reaction between A and TG cancompete with A-SH molecules for the limited number of Y functions on B,there are certain constraints on the range of conjugation conditionsthat can be employed. The initial purity of reagents, especially of TG,is an important consideration as this is a potential source of unwantedfree thiols (e.g. hydrolysis products of TG). Increasing the pH of theconjugation reaction will tend to deprotonate amines on A, resulting ina faster reaction of amines with TG, but the rate of hydrolysis of TGmay increase too. The efficiency of conjugation will therefore depend,as with any chemical reaction, on the concentrations of reactants, butalso on the initial level of thiol contamination, the rate of productionof A-SH (from a reaction of A with TG) compared with that of otherthiols, the relative reactivity of the different thiol-containingmolecules with B-Y, and the total amount of Y functions available forconjugation.

The inventor has discovered that conjugations of molecules of theformula B-Y_(n) (where ‘n’ is an integer) with A-SH will become moresusceptible, to interference from contaminating thiols as ‘n’ becomessmaller. If n=1, the reaction of a molecule of B-Y with just onemolecule of contaminating free thiol will prevent that particular B-Ymolecule from participating in any other coupling reactions. Thus, if‘n’ is small, a large excess of B-Y may be required to ensure that eachA-SH molecule can react with B-Y. The use of excess B-Y may beimpractical and uneconomic, particularly if B is a large biomolecule.Moreover, high levels of free B in the final conjugate may betroublesome in certain applications. Instead the inventor realised thatan improved method of preparing conjugates in the face of undesirablecompeting reactions with unwanted thiols was to increase efficiency ofthe conjugation between A-SH and B-Y_(n) by using B-Y reagents with highvalues of ‘n’.

The method of the present invention becomes increasingly robust as thevalue of ‘n’ increases. Preferably the second chemical entity comprisesmore than three sulfhydryl-reactive groups per molecule. More preferablythe second chemical entity comprises five or more sulfhydryl-reactivegroups per molecule. Most preferably the second chemical entitycomprises ten or more sulfhydryl-reactive groups. Advantageously thesecond chemical entity comprises from ten to fifteen sulfhydryl-reactivegroups. If the desired number of sulfhydryl-reactive groups are notnaturally inherent in the second chemical entity, they may be introducedby chemical or enzymatic synthesis, as described elsewhere. For presentpurposes, a “sulfhydryl-reactive group” is one which will react with asulfhydryl group under the conjugation reaction conditions. Clearly thereaction conditions must be such as to substantially preserve theactivity of the first and second chemical entities.

In the case of HRP, which has only six lysine residues, only two ofwhich can be exploited for the introduction of Y functions (BioconjugateTechniques 1996, G T Hermanson, p 632), it is particularly importantthat reagents have low thiol content and that the reaction conditions donot lead to excessive hydrolysis of TG. However, irrespective of theconditions used, any reduction in conjugation efficiency arising fromthe production of unwanted free thiols may be countered by introducingmore Y groups into B or by polymerising B-Y, or by a combination ofthese two approaches. The method used to introduce extra Y groups into Bis not particularly limited, though the method should preferablysubstantially preserve the biological activity of B, particularly if Bis an enzyme.

An advantage of the present invention is that it avoids the need toemploy large molar ratios of second chemical entity to thiol generator(i.e. B:TG ratio) to overcome the presence of interfering free thiols.Thus, in the present invention, the molar ratio of second chemicalentity to thiol generator in the conjugation reaction is convenientlynot more than 2.0:1, typically 1:1 or less, preferably in the range 1:1to 1:20, most preferably in the range 1:10 to 1:15.

Y groups can be directly attached to functional groups that are alreadypresent on B. Alternatively, or in addition, new reactive centres may beintroduced. For example, molecules with sugar chains may be oxidisedwith sodium periodate to generate aldehyde functions. These functionsreadily react with amine- or hydrazide-containing compounds to formSchiff bases or hydrazone linkages, respectively, which can bestabilised with sodium cyanoborohydride, sodium borohydride or anothersuitable reducing agent. Thus if an excess of a diamine compound isreacted with aldehyde groups, one amine moiety will be introduced foreach aldehyde that is modified. If the diamine is not present in excesscross-linking reactions (i.e. to give B polymer) may occur, which may beuseful in certain situations (see below).

The introduction of amine groups into B is not limited to the reactionof B with simple diamines. Other molecules with two (or more) functionalgroups may be employed. One of these functional groups must be able toreact with B and it must be possible to convert the other functionalgroup into a Y group. Y groups may also be introduced directly into Bwithout utilizing any amine moieties on B. For example, if B is aglycoprotein, reaction with periodate generates aldehyde functions whichmay subsequently be reacted with a heterobifunctional reagent that hasboth aldehyde and sulfhydryl reactive groups (e.g.4-(4-N-Maleimidophenyl) butyric acid hydrazide; MPBH) (Chamow S M et alJ. Biol. Chem. 1992 267, 15916-22). Compounds analogous to MPBH (e.g.M₂C₂H) may also be used (Bioconjugate Techniques 1996, G T Hermanson, p250).

Another method of introducing functional groups is to modify carboxylicacids (e.g. as provided by glutamate or aspartate residues inpolypeptide chains). Carbodiimide-mediated condensation ofamine-containing molecules with carboxylic acids is widely employed inchemical synthesis and can be used to introduce amines (e.g. by reactionwith diamines, triamines) and other functional groups into B. Forexample, aminated HRP has been generated using carbodimide(EDC)-mediated condensation of HRP with ethylenediamine (U.S. Pat. No.5,039,607), which gave HRP molecules with 11 amine functions. Thecarbodiimide approach is particularly useful if periodate-based methodscannot be employed through lack of sugar chains on B.

One advantage of introducing amines into B followed by subsequentconversion into Y functions is that a wide range of potentially usefulamine-containing molecules is commercially available. In thesemolecules, the amine groups may or may not be attached to the same atom.By increasing on B the number of amines (which may ultimately convertedinto Y groups), derivatives of HRP that are more resistant to thiolinterference in conjugation reactions may be generated.

In a preferred embodiment, periodate-activated HRP is reacted with amolecule C which bears ‘c’ amine groups (where c=2 or more), providingHRP analogues with c−1 additional amines for each aldehyde groupmodified. In preferred embodiment C is a diamine (e.g. ethylene diamine,propane diamine, butane diamine; 2,2′(ethylenedioxy)bis-ethylamine(EDBA); lysine and the like) or molecules with three or more aminefunctions (e.g. lys-lys (c=3), trilysine (c=4), Jeffamine T403 (c=3),aminated dextrans, aminated dendrimers and other polyamino species.

An alternative strategy to reduce the impact on conjugation efficiencyof unwanted free thiols is to induce polymerisation of B-Y, whichprovides a polymer [(B)n]q with nq maleimide functions, where ‘n’ is thenumber of Y functions per molecule of B in the polymer, and ‘q’ is theaverage number of B molecules in a polymer. Thus even in situationswhere ‘n’ is small, the impact of free thiols is reduced because the Bmolecules are physically connected and conjugation of A-SH to any one ofthe available Y functions effectively tethers all B molecules in thepolymer to A. Another advantage of this approach is that the sensitivityof detection might be increased as a larger number of, for example, HRPmolecules potentially can be attached to A. The use of polymeric HRP inimmunoassays to increase assay sensitivity is well known and such formsare commercially available.

Alternatively, B molecules (rather than B-Y) with free amines (eitheroccurring naturally or introduced for example by reaction with diamines)may first be polymerised by reaction with homobifunctional crosslinkingagents (e.g. dialdehydes) or by use of heterobifunctional reagents topromote coupling of amines to other functional groups on B.Heteropolymers may also be generated by reaction of B with ‘scaffold’molecules (e.g. dendrimers, dextrans, proteins) to which multiple Bmolecules may be appended. This may involve reactions of amines on Bwith aldehydes on the scaffold or with other available functional groups(e.g. thiols) mediated by heterobifunctional crosslinking reagents. Inturn, remaining unreacted surface amine functions can be converted intoY groups by reaction with a heterobifunctional reagent such as SMCC. Ifinsufficient free amines remain after the polymerization step, furtheramines can be appended prior to introduction of Y groups. For example,if carbodiimide chemistry is used to introduce amines into B which arethen utilized, or partly utilized, in polymerization reactions, adifferent chemistry (e.g. periodate activation of sugar chains) may beused to introduce additional amines (which can then be converted into Ygroups) or to introduce Y groups directly (e.g. by reaction with MPBH).Polymerised HRP may also be obtained from commercial sources and furthermodified to create multiple Y functions for use in the presentinvention.

B molecules that confer useful properties on conjugates are oftenprepared and stored in B-Y form for later use. For example, some labelsare available commercially as maleimide-activated derivatives(lyophilised maleimide-activated enzymes, or maleimide or iodoacetylderivatives of small fluorescent molecules). B-Y may also be freshlyprepared if required using methods known in the art. Examples of Binclude enzymes such as horseradish peroxidase (HRP), alkalinephosphatase, and glucose oxidase (Gox); fluorescent molecules such asphycobiliproteins (e.g. allophycocyanin, phycoerythrin), low molecularweight dyes (e.g. fluorescein, rhodamine) and the like. Bridging orlinker molecules include streptavidin or biotin. Cell killing agentsinclude toxins (e.g. ricin, and radioisotopes). The enzymes HRP andalkaline phosphatase are especially widely-used enzyme labels forantibodies and other polypeptides, and these enzymes represent preferredexamples of the second chemical entity.

When practising the invention A, TG and B-Y are combined and incubatedfor suitable period of time. The order of addition may be varied to suitcircumstances.

For example, in one approach in a first step TG is mixed with A. After asuitable period of time during which A-SH is generated, in a second stepthe mixture is added to B-Y. A suitable period of time is any time thataffords an efficient conjugation between A and B-Y. The period istypically up to 18 hours (i.e. overnight), but may be performed withinabout 2-4 hrs under optimum conditions. The reaction conditions in thetwo reaction steps may be varied if appropriate, for example the firstand second steps may be carried out at different pH values by use ofappropriate buffers.

In another approach, A is mixed with B-Y after which the mixture iscontacted with TG.

In a further approach, TG is mixed with B-Y and the mixture is thencontacted with A.

Desirably, either one or both of TG and B-Y are initially in driedcondition e.g. being freeze-dried or lyophilised for storage stability.Preferably, a solution (typically aqueous) of the first entity is usedto reconstitute the other components, which leads to minimal expansionof the sample volume.

In a particularly preferred embodiment, A is added in liquid form to alyophilised mixture comprising both B-Y and TG.

Alternatively, a lyophilised mixture of TG and B-Y is reconstituted witha solvent (typically water) to give a mixture not containing A, to whichA is subsequently added.

The solution in which the conjugation reaction takes place mayoptionally include one or more components in addition to A, B-Y and TG.These components may or may not affect the rate of the conjugationreaction. For example, some additives might be introduced prior tolyophilisation of components, primarily for the purpose of stabilisingsaid components or for ease of dissolution. Other components, especiallybuffers, may be employed primarily to provide conditions of pH underwhich the preferred reactions take place at a suitable rate. Thesebuffers may be introduced into the final conjugation mixture via one ormore additions such that the final mixture has the required composition.Preferably, though not obligatorily, the buffering substances areincluded along with one or more of the other components (A, B-Y or TG,or a combination thereof), to minimise the amount of labour in preparingthe conjugate.

The stability of TG and the conditions required to effect conjugationshould be carefully considered when determining the preferred order ofadding components to the reaction mixture. For example, base labile TGmight be stored in acidic medium (or lyophilised from such medium) andintroduced last into a suitably buffered reaction mixture. The bufferedmixture can be designed to accommodate the acid introduced along with TGand provide a final pH and composition that is suitable for the intendedconjugation reaction to take place.

Suitable conditions of, for example, temperature, pH and concentrationfor the conjugation reaction will depend upon the nature of thereagents. Suitable conditions can readily be determined by those skilledin the art by means of routine trial and error.

The method of the present invention also provides the basis of aconjugation kit. TG and B-Y are provided preferably as dried, e.g.freeze-dried (lyophilised) components, either separately or as amixture, in suitable vessels, along optionally with a suitable buffer inwhich A can be dissolved. Alternatively, A can be desalted or dialysedinto the said buffer, particularly if the formulation of A containscomponents that might interfere with the conjugation reaction.

In a further aspect the invention thus provides a conjugation kit foruse in the method of the invention, comprising at least one sample ofreagent selected from a first chemical entity, a second chemical entityand a thiol generator, and instructions for performing the method of theinvention.

The kit desirably comprises samples of at least two reagents selectedfrom a first chemical entity, a second chemical entity and a thiolgenerator.

The kit preferably comprises a sample of a second chemical entity and asample of a thiol generator. These two reagents may be providedseparately or may be in the form of a mixture. The thiol generator ispreferably present in excess in relation to the second chemical entity,e.g. up to about 20 times molar excess.

A plurality of aliquots or samples of reagents or reagent mixtures arepreferably provided in suitable containers, e.g. in individual tubes,vials or in the wells of a multi-well (e.g. 96 well) microtitre plate.Samples may be provided in a range of different predetermined amounts,so a user can select the appropriate sample size having regard to thematerial (e.g. first component) to be treated. One preferred embodimentcomprises a plurality of samples of mixtures of second chemical entityand thiol generator in a range of different amounts for use inconjugating one or more a different first chemical entities (possiblysupplied by an end user) e.g. for labelling a range of differentmolecules, for instance antibodies to be used in direct immunoassays.

The samples of reagent or reagent mixtures may optionally include othermaterials such as buffers etc. to provide appropriate conditions forreaction.

Where the thiol generator comprises 2IT, the samples of 2IT arepreferably at a pH below 8, more preferably below 7.8 and yet morepreferably below 7.7. A preferred range of pH is 7.0-7.5.

The kit may include optional ingredients such as solvent, buffersolutions etc.

The reagent samples are desirably in dried, e.g. freeze dried(lyophilised) form for storage stability. Desirably the reagent samplesare freeze dried from an aqueous solution comprising sodium phosphatebuffer at a pH in the range 5-6.5, preferably 5.0-6.0. Desirably alsothe reagent samples are freeze dried from an aqueous solution comprisingMg²⁺ ions, especially at a concentration in the range 1-10 mM Mg²⁺.Conventional cryoprotectants and lyoprotectants may also be present,such as polyols, especially trehalose or dextran.

In a preferred embodiment, lyophilised reagents are provided in smallpolypropylene tubes (e.g. 0.5 ml or 1.5 ml Eppendorf tubes),polypropylene cryovials or vials, glass vials, 96-well polypropylene orpolystyrene microplates, and other receptacles appropriately sized forthe intended conjugation reaction. Preferably, the material of thevessel does not significantly react with TG to release thiol groupseither before lyophilisation or upon subsequent reconstitution with asuitable solvent. The nature of the vessel is not particularly limitedto the aforementioned examples.

Suitable buffering components for use in the present invention includephosphate buffers, especially sodium phosphate,N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonicacid (MOPS), bicarbonate and other buffers that do not react with TG, orreact relatively slowly when compared with the rate of reaction of TGwith functional groups on A. The list may therefore containamine-containing buffers that react at a suitably slow rate.

Other components of the conjugation reaction mix may include salts (e.g.NaCl) and other inorganic or organic components that do not directlyparticipate in the reactions but provide a suitable environment thatstabilises components or in some other way facilitate the desiredreactions or minimise losses, for example, on the surfaces of vessels.

Since TG is reactive it may react with other nucleophiles in theconjugation mixture. Water is a weak nucleophile but it is present at ahigh concentration and hydrolysis reactions could increase theconcentration of thiols not covalently associated with A, especially atpH values significantly above pH 7.

Preferably the TG includes little or no free thiol groups, with thelevel suitably being below 5% in molar terms, preferably below 3% inmolar terms and more preferably below 1% in molar terms.

TG from commercial sources may contain significant quantities of freethiol, and free thiols may also be generated over a period of time instorage. Free thiols could compete with A-SH for Y groups on B-Y andreduce conjugation efficiency. In the case of 2-iminothiolane, onesupplier states that contamination with free thiols is ‘up to 5%’. Thebatches used for work described here were measured at about 1% thiolcontent in molar terms.

It is preferred that the molar ratios of the reactants are carefullyselected so that small quantities of free thiols possibly present in TGdo not impact significantly on conjugation efficiency. Traut's reagentis more stable than other molecules that are used to introduce thiols orprotected thiols and it is not necessary to use large molar excess. Someamine-reactive heterobifunctional reagents with NHS groups have shorthalf-lives in aqueous solutions and are used in large excess tocompensate for rapid hydrolysis. Typically, TG is used in reasonableexcess, e.g. 10 times molar excess over relevant chemical functionalitysuch as amines present on the first chemical entity to be sure that allmolecules of the first chemical entity are thiolated. However, inselecting a suitable concentration of TG the user must have regard forthe likely rate of reaction, which is influenced by the pH of thesolution. A suitable concentration of TG at a fixed pH is readilydetermined by examining the effect of varying the concentration of TG onthe performance of the resulting conjugates. It is preferred that thereaction conditions allow efficient thiolation of A, but that excessivethiolation is avoided so as not to damage the biological activity of A.Equally, excessive amounts of the second chemical entity should not beconjugated to A-SH otherwise this might lead to suboptimal performanceof the conjugate. The second chemical entity is typically present inmodest excess, e.g. up to about 5 times molar excess, in relation to thethiolated first chemical entity, to be sure all of the first chemicalentities are conjugated. After reaction, excess materials can be removedby any suitable techniques.

Using methods taught in the present invention a conjugation reactionmight contain just 10 μg of an IgG antibody (Mr 150,000). Typicallyabout 5 thiol groups per molecule are introduced. Thus in a 10 μl volume(6.7 μM antibody) the concentration of amines (to be modified) is ˜33μM. Since there is no desalting step, nascent thiols will reactimmediately with B-Y. For a label such as maleimide-activated HRP(molecular weight 40,000) the labelling of all nascent thiols wouldrequire ˜33 μM enzyme, which corresponds to ˜13 μg of HRP in a 10 μlreaction volume. If a 25-fold molar excess of TG were used, 1% thiolcontamination in the solution of TG would represent a concentration ofabout 8 μM, or ¼ (in molar terms) of the maleimide-activated HRPpresent. Moreover, if HRP were labelled on average with 2 or more Ygroups per molecule, the reaction of an HRP molecule with onecontaminating free thiol would not necessarily prevent that moleculefrom conjugating with B-Y. It follows from this that the use of excessB-Y, or B-Y with multiple Y groups, might ameliorate the negativeeffects of contaminating free thiols and those generated by hydrolysisof TG during the conjugation reaction. In some applications, especiallythose with antigen immobilised on a surface, the use of excess B-Y isnot problematic as the surplus reagent (i.e. not linked to A) can simplybe washed away during the immunoassay. Moreover, it is common to useexcess B-Y to minimise the amount of unreacted A-SH, which would competewith AB and reduce assay sensitivity.

In some immunoassay applications, especially those in which the antigenis measured in free solution, it may be desirable to maximise the amountof conjugated B and minimise free B-Y. This can be achieved bypurification of AB from the conjugation mix using methods known in theart or by careful control of the reaction conditions. In the presentinvention, the avoidance of desalting steps, from which the yield ofmaterials is difficult to determine, especially when there is a need toprogress quickly to the conjugation step, greatly assists inestablishing precise ratios of the reactants and in optimising theconditions to meet specific experimental objectives.

The level of free thiols becomes more important if the concentration offree B-Y needs to be kept low, since unwanted thiols might render B, andespecially B that is lightly decorated with Y (that is, the value of nin B-Y, is low, e.g. at or near 1), incapable of conjugating to A-SH.Where separation of free B-Y following conjugation is difficult orundesirable, it is preferable to minimise side reactions by usingconditions (i.e. low pH, amine-free buffers) that do not causenon-A-dependent thiol release.

Free thiols may be removed or largely removed before TG is used for thepurpose of thiolating A. For example, free thiols may be removed bypurification using a solid support to which TG and the free thiol formshow different affinities, thus allowing for the selective elution ofrelatively pure TG. The binding to the solid support may be covalent ornon-covalent.

In one approach a solution of TG is contacted with a solid support towhich Y groups have been attached. Such materials are commerciallyavailable, as in the form of iodoacetyl Sepharose (Pierce) (Sepharose isa Trade Mark) or can be made using methods known in the art (J. Biol.Chem. 245 3059-3065 (1970) by carbodiimide-mediated conjugation ofhaloacetates to amine-bearing agarose beads. In a preferred embodimentthe number of Y groups exceeds the number of free thiols such that thesolid support is able to capture all, or substantially all, of theunwanted free thiols via a covalent bond. The TG not binding to thesolid support preferably is used immediately or quickly frozen andlyophilised to preserve the material largely in an intact state.

Another approach is to measure the concentration of free thiol insamples of TG e.g. using the well-known 5,5′-dithiobis(2-nitrobenzoate)(DTNB) method, or other suitable method for measuring free thiols. Asolution containing a Y-bearing molecule and lacking functional groupsthat might interfere with subsequent conjugation steps is then added,preferably in slight molar excess and preferably under conditions inwhich (a) further thiols are not generated or are generated very slowly(b) the contaminating thiols react quickly with Y to form a stablethioether, thus eliminating thiols from the sample. This strategy can ofcourse be used to remove thiols regardless of their source.

The Y-bearing molecule suitably contacts TG for a period of time thatallows most of the free thiol to react with Y. After the said period oftime, the TG sample is contacted with A and B-Y. The molecule used toremove free thiols may be one of the following: N-ethylmaleimide,iodoacetamide, iodoacetate, bromoacetamide, bromoacetate, chloroacetate,mercurial compounds and the like. N-ethylmaleimide is particularlypreferred.

By contacting A, TG and B-Y at the same time, in accordance with theinvention, TG may be afforded the opportunity to react with amines thatare present on B-Y, as well as A. If B is a large biomolecule it isquite likely that free amines will still be present, even if Y groupshave already been introduced via chemical modification of availableamines. In the case of HRP, a very popular label in bioconjugatechemistry, the number of amines per molecule is unusually low (0.15 perkDa), compared with 0.88/kDa for bovine serum albumin (BSA) and 0.44/kDafor ovalbumin. Thus the degree of reaction of TG with B-Y can becontrolled by varying (i) the type of B used (ii) the density of Ygroups on B (with respect to residual free amines) (iii) the order ofaddition of reagents.

Where the level of amines on B is high and, under the experimentalconditions used, a significant reaction with TG occurs, B might bepartially polymerised (i.e. reaction of B-Y with nascent B-SH). Wherethis is considered undesirable, simply adopting a two-step conjugationstrategy will circumvent the problem. First, TG contacts A and after asuitable period of time in which A-SH is generated the mixture iscontacted with B-Y. This allows AB conjugates to be formed beforesignificant amounts of B polymer can be generated.

Partial polymerisation of B-Y may be advantageous in some situations.One simple approach to increase immunoassay sensitivity is to attachmore B per molecule of A. Polymerisation of B-Y has been used to achievethis goal. By careful selection of reagent concentrations (TG, A) andthe duration of the first step (A-SH production) and consideration ofthe number of free amines and Y groups on B, the extent of in situ Bpolymer production can be manipulated as required.

While the one-step conjugation method of the present invention isexceptionally attractive, with all reagents combined together in asingle step, two-step variants (e.g. involving two reagent additionoperations instead of one) are still remarkably simple compared withmost present conjugation methods, and have the benefit of providingfurther options, if required, to optimise conjugates for specificapplications.

For example, in one embodiment, a reaction is set up under conditionsthat favour the production of AB conjugates (from A-SH and B-Y). In aseparate reaction, B is reacted with TG to generate a solutioncontaining B-SH, which is then added to pre-formed AB complexes, whichmay contain unreacted Y groups. By introducing B-SH, a controlledincrease in molecular weight of conjugate can be obtained through acoating of B-SH on AB-Y molecules. By careful consideration of the inputof B-Y and the density of Y groups, the initial conjugate can beengineered to react to varying extents with molecules of B-SH.

Some applications may require conjugates of relatively low molecularsize. Applications that involve penetration of reagents into anantigen-containing sample (e.g. immunohistochemistry) will normally workbest with low molecular weight conjugates. However, if the antigen isdeposited and exposed on a surface (e.g. nitrocellulose, as in westernblotting) it may show greater sensitivity with a higher molecularconjugate. Thus a consideration of the intended use determines thepreferred approach for developing the conjugate.

In the preparation of immunoconjugates using thiol-based strategies itis common to employ a ‘blocking’ step at the end of the conjugationreaction to remove any unused thiols. In some cases this step is notactually required but is performed as a matter of routine. In thepresent invention there is unlikely to be any need for a blocking stepif TG is 2-iminothiolane as the thiols involved in coupling areself-limiting because of the secondary intramolecular reaction. However,since the ring-opening reaction must precede any decay via the secondaryintramolecular reaction, or via conventional thiol blocking strategies,a simple deactivation step may be utilised to accelerate ring opening ofexcess TG.

The quickest method for halting conjugation and deactivating TG is toadd a nucleophile (Nu), e.g. glycine, in a suitable buffer. The rate ofdeactivation is a function of pH and type and concentration of Nu; theconditions employed to deactivate TG must be compatible with theconjugate and preferably the application in which the conjugate will beused, otherwise a purification step will be required. Thiols releasedfrom TG in this way will react intramolecularly or with excess Y groupson B-Y or AB-Y. Thus the addition of Nu can deactivate both TG and,indirectly, Y groups. Since the released thiols may form a covalent linkto AB the choice of Nu needs to be carefully considered so as not tointroduce unwanted groups derived from Nu (e.g. bulky substituents) tothe conjugate. However, treatment of the mixture with a low molecularweight thiol (e.g. mercaptoethanol) prior to ring opening of TG may becarried out to deactivate Y groups if required.

The conjugation reactions are conveniently terminated by the addition ofNu e.g. glycine to attack excess TG and also by addition of athiol-blocking reagent (TBR), such as N-ethylmaleimide, which iscommonly employed in bioconjugate chemistry for this specific purpose.However, the combination of glycine as Nu and N-ethylmaleimide as TBR isnot meant to be limiting and many other possible Nu materials and thiolblockers will be apparent to one skilled in the art.

In a preferred embodiment Nu and TBR are introduced sequentially.Preferably, Nu is added before TBR. Preferably TBR is added in slightexcess over released free thiols. The level of free thiol can bedetermined, for example, with DTNB. Where the level of thiol cannot bemeasured, TBR is added in slight excess over the known level of TG,since the level of thiol cannot exceed the initial concentration of TG,if TG is the only source of free thiol.

In one embodiment, the conjugate mixture is simply contacted with Nu(e.g. 50 mM glycine in phosphate buffer or phosphate buffered saline(PBS), pH 8.0). Optionally, the mixture may be further supplemented withthiol blocking reagents (e.g. N-ethyl maleimide), stabilisers (e.g. BSA,ovalbumin or other proteinaceous components; glycerol) andanti-microbial reagents (e.g. sodium azide) or other preservatives.Conjugate may be stored at 4° C., or in small aliquots frozen at −20°C., or in liquid form at −20 (i.e. with 50% glycerol) or frozen at −70°C., depending on the nature of the conjugate and its temperaturestability and sensitivity to freeze-thaw.

One area of application of the present invention is in the production ofconjugates for immunisation. A significant number of primary antibodyreagents are prepared against small peptides, which are usuallyconjugated to a carrier protein. Suitable carriers includemaleimide-activated keyhole limpet haemocyanin (KLH), BSA, ovalbumin,and the like. For peptide antigens that have not been chemicallysynthesised with terminal cysteine residues, and for other smallmolecules containing only amines, the present invention allows thegeneration in situ of thiol functions for coupling to e.g. amaleimide-activated carrier. The avoidance of desalting steps issignificant here, not simply because the procedure is more convenient,but also because for molecules of low molecular weight purificationfollowing thiolation is often difficult or impossible. In suchapplications the presence of excess B is not problematic and in molarterms B can greatly exceed A, to ensure that there is excess nascentthiol to couple to all maleimide functions on the carrier.

Another application of bioconjugates is in immunotoxin therapy, whichinvolves antibody-mediated delivery of substances that can kill specificcell types (e.g. cells expressing antigens that are diagnostic of thecancerous state). Some of these toxins are extremely dangerous andrepresent significant hazard to those engaged in the production oftherapeutic agents. The inherent simplicity of the present inventionaffords the opportunity to conjugate powerful toxins to antibodieswithout generating dangerous liquid waste from desalting columns ordialysis steps. For example, samples of ricin might be contained in asuitable vessel and the entire conjugation procedure carried out in thatsame vessel. In this situation, the antibody preferably is maleimideactivated and combined with the toxin, which is treated in situ with TGto release thiols for the conjugation reaction.

Another application relates to labelling with B molecules that includefluorescent amine-containing small molecules. Mostly, fluorescent dyesare NHS-activated and are reacted with amines on A. Such reagents arevery unstable in aqueous solutions and deteriorate rapidly if not storedin a dry state. Consequently, the preferred approach often is to useexcess reagent followed by purification of AB from B. However, analternative approach is to mix a stable amine-containing fluorophorewith a lyophilised maleimide-activated carrier and generate a reactivethiol in situ using TG. In this way excess highly unstable fluorophoreis not required and the dye does not contain a reactive group that canbe hydrolysed upon storage.

The methods of the present invention teach how conjugates may be madeusing proven heterobifunctional chemistry but without the troublesomedesalting steps that increase labour and limit its application tocomponents that are available in relatively large quantities. Multiplethiolation experiments can be performed using tiny amounts of materialand conjugate performance can be optimised rapidly and cost-effectively.The methods described in the present invention lend themselves readilyto automation. For example, exploration of 96 different thiolationconditions in a microtitre plate would be considered straightforwardusing present robot technology. Optimisation of thiolation procedures onthis scale would be completely impractical in processes involvingdesalting or dialysis steps.

One of the major applications of the present invention is labelling ofprimary antibodies. Since existing antibody reagents may have beenformulated without consideration of direct labelling, a number ofadditives include anti-microbial agents such as sodium azide, andstabilisers such as BSA or glycerol might be present. Furthermore, theantibody storage buffer may not be one of the preferred buffers forconjugation reactions. In other cases, the antibody might be provided asa crude sample of frozen serum or ascites fluid.

For samples that contain additives such as BSA or other proteins derivedfrom animal fluids, including substances from tissue culture processes(e.g. foetal calf serum) the antibody may be a minor component anddifficult to label selectively. Fortunately, methods for purifyingantibodies are well known, and include biospecific affinitychromatography on a support matrix to which the antigen has beenattached. Other suitable methods include support matrices with coupledprotein A, protein G, and the like, which can be used to purify IgG fromcomplex mixtures by exploiting interactions with the Fc regions ofmammalian IgGs. The elution buffer should be carefully selected tofacilitate subsequent conjugation reactions. For example, in affinitychromatography elution is often performed using low pH buffer (e.g.glycine pH 2.3), which would not be suitable, at least not if thematerial were to be added directly to the conjugation mixture. Other lowpH buffers those are more preferable include citrate/citric acid andthose based on HCl/NaCl mixtures.

Another approach for removing substances such as BSA is chromatographyon a resin such as Blue-Sepharose (Amersham). The advantage of thisapproach is that the antibody passes through the resin and is notexposed to low pH treatment, which could damage some antibodies. A newproduct Melon (Melon is a Trade Mark) gel from Pierce has recently beenmade commercially available, which apparently removes a broad range ofproteins from antibody samples. However, subtractive methods such asthese do not remove unwanted low molecular weight substances.

Methods that involve purification of the antibody of interest by bindingto an antigen or protein on a support matrix have the advantage that allunwanted molecules are washed away. Where low pH elution cannot be usedto disrupt the antibody:antigen interaction, perhaps because of risk ofdamage to one of the components, an alternative elution strategy, suchas hypotonic elution (e.g. Gee & Kenny, Biochem J. 230, 753-764, 1985)can be used.

A variety of applications have been exemplified, and show that theinvention will allow primary antibodies and other reagents, which aregenerally expensive and available to researchers in small quantities, tobe labelled easily. This is likely to result in far greater use ofdirect detection methods, which have a number of advantages over otherindirect methods of antigen detection.

For the avoidance of doubt it is hereby explicitly stated that anyfeature described herein as “preferred”, “desirable”, “convenient”,“advantageous” or the like may be employed in the invention inisolation, or in combination with any one or more other features sodescribed, unless the context dictates otherwise.

The invention is further described, by way of illustration, in thefollowing Examples which refer to the accompanying figures in which:

FIG. 1 is a bar chart of absorbancy versus concentration of 2IT showingresults of ELISA of Ab1-HRP conjugates as tested in Example 4;

FIG. 2 is a graph of absorbancy versus pH showing the effect of varyingpH on conjugation efficiency as tested in Example 5;

FIG. 3 is a graph of absorbancy versus pH showing pH optimum for Ab1-GOXconjugation as tested in Example 6;

FIG. 4 is a bar chart of absorbancy for various different buffersshowing the effect of buffer type on conjugation efficiency as tested inExample 7;

FIG. 5 is a pair of graphs of absorbancy versus pH for phosphate bufferand Tris buffer as tested in Example 8;

FIG. 6 is a graph of absorbancy versus time showing the time course ofconjugate formation as tested in Example 9;

FIG. 7 is a bar chart of absorbancy for different samples showing theeffect of varying the order of addition of reagents as tested in Example10;

FIG. 8 is a graph of absorbancy (arbitrary units, 405 nm) againstconjugate dilution (log scale), showing performance in an ELISA of twodifferent batches of EDBA-HRP-IgG conjugates (solid circles and emptycircles) compared with that of an OLA-HRP-IgG conjugate (squares).Control data generated with an antigen-free microtitre plate aresuperimposed on the baseline.

FIG. 9 is a graph of absorbancy (arbitrary units, 405 nm) againstconjugate dilution (log scale) showing the performance in an ELISA ofEDBA-HRP-Ig conjugates prepared at pH 6.5 (open circles), pH 7.0 (solidcircles), pH 7.5 (squares) or pH8.0 (triangles).

FIG. 10 is a graph of absorbancy (arbitrary units, 405 nm) againstconjugate dilution, (log scale) showing the performance in an ELISA ofan EDBA-Gox-Ig conjugate (solid circles) and an OLA-Gox-Ig conjugate(open circles).

FIG. 11 is a graph of absorbancy (arbitrary units, 405 nm) againstconjugate dilution (log scale), showing the performance in an ELISA ofan iodoacetyl-activated EDBA-HRP-Ig conjugate (data for controlsuperimposed on the baseline).

FIG. 12 is a bar chart of absorbancy (405 mm) against ratio by weight ofIg to mal-EBDA-HRP (solid black bars) or mal-OLA-HRP (shaded grey bars).

FIG. 13 is graph of absorbancy versus time showing the release of thiolfrom 2IT by various amines as tested in Example 17;

FIG. 14 is a graph similar to FIG. 13 showing thiol capture using DTNBas tested in Example 18; and

FIG. 15 is a graph of absorbancy (405 nm) against conjugate dilutionfactor, showing ability of freeze-dried reagents to form conjugateactive in ELISA after storage at different temperatures.

EXAMPLES Example 1

HRP (5 mg) (Sigma, P6782) (B) in 0.5 ml of 100 mM sodium phosphate, pH7.2, was activated with sulfo-SMCC (4 mM) (Pierce, 22322) for 1 hour at25° C. The maleimide-activated HRP (‘mal-HRP’) (B-Y) was desalted on aPD10 column (Amersham Biosciences) equilibrated with 10 mM sodiumphosphate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH7.03. The activated protein (2.5 mg/ml) was used either immediately orlyophilised.

Example 2

Glucose oxidase (5 mg) (Biozyme GO3B3) in 0.5 ml of 100 mM sodiumphosphate, pH 7.2, was activated with sulfo-SMCC (2 mM) for 30 min at25° C. The maleimide-activated Gox (mal-Gox) was desalted and processedas described in Example 1.

Example 3

Rabbit IgG (Sigma I5006) was dissolved at 1 mg/ml in Tris bufferedsaline (TBS) (50 mM Tris/150 mM NaCl, pH 8.0) and stored in smallaliquots at −70° C. To prepare coated ELISA plates, the IgG was thawedand diluted in TBS to 20 μg per ml. Nunc maxisorb plates (clear,96-well) (code 071832) were incubated with 50 μl (1 μg) of IgG per well.Plates were coated for >1 hour at room temperature and then wrapped infoil and transferred to 4° C. for storage. Coated plates were usedwithin 10 days. Immediately before use, plates were blocked withTBS/0.1% BSA, pH 8.0, for >30 min. To test conjugates by ELISA,duplicate or triplicate wells were incubated with 50 μl of conjugatesuitably diluted in TBS/0.1% BSA. After 60 minutes at 25° C., plateswere washed five times with TBS. A suitable substrate (see below) wasadded and absorbance was determined after 2 or 10 minutes at anappropriate wavelength (depending on the label used) using a Victor3model 1420 multi-label counter (Perkin Elmer). HRP activity was measuredusing 1 mM 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) substrate in 50 mM sodium acetate, pH 5.0, containing 1 μl H₂O₂per ml of reagent. Gox activity was measured in a coupled assay systemusing 100 mM sodium acetate, 100 mM glucose, pH 5.0, containing HRP (50μg/ml) and 2 mM ABTS. Alkaline phosphatase activity was measured using 5mM para-nitrophenyl phosphate (PNPP) in 50 mM glycine buffer, pH 9.6,containing 1 mM MgCl₂ and 0.5 mM ZnSO₄.

Example 4

Lyophilised anti-rabbit IgG antibody (1 mg) (Sigma R2004) (‘Ab1’) wasresuspended in 1 ml of 150 mM NaCl and stored at 4° C. The followingstocks of 2-iminothiolane (2IT) in 1.2 mM HCl were prepared: 100 mM, 100mM, 10 mM, 1 mM, 0.1 mM and 0.01 mM. Ab1 (A), mal-HRP (B-Y) (prepared asdescribed in Example 1) and buffer (100 mM sodium phosphate, 1 mM EDTA,pH 7.4) were combined in a 1:1:2 ratio and 20 μl portions were dispensedinto 1.5 ml Eppendorf tubes. Tubes received 5 μl of 2IT (TG) to givefinal 2IT concentrations of 200 mM (C7), 20 mM (C8), 2 mM (C9), 0.2 mM(C10), 0.02 mM (C11) and 0.002 mM (C12). A control tube (neg) received1.2 mM HCl instead of 2IT. ‘Pos’ is a positive control antibody, diluted1/1000 (Sigma A6667). After 90 minutes, samples C7-C12 were diluted withTBS/0.1% BSA and tested by ELISA using the procedure of Example 3 at adilution (with respect to undiluted Ab1) of 1/200. Results are shown inFIG. 1.

As can be seen in FIG. 1 there is a bell-shaped dependence of absorbancyversus 2IT concentration. At low concentrations of 2IT this effect isprobably explained by insufficient thiolation of Ab1 to allow efficientconjugation. At high concentrations, the effect is probably explained bydamage of Ab1 through excessive modification of lysine groups, thoughother explanations are possible. For example, commercially available 2ITis contaminated with a small percentage of free thiols, which couldcompete with thiolated Ab1 for reaction with mal-HRP at highconcentrations of 2IT. There is little absorbancy in the absence of 2IT(neg). Absorbancy values for control wells were low (<0.1) except for C7(^(˜)0.25). The data obtained for control wells (with no coated antigen)were subtracted from data obtained for antigen-coated wells to give thevalues shown in FIG. 1. This experiment shows that it is possible tocombine 2IT, Ab1 and mal-HRP in a single tube and generate activeconjugates.

Example 5

The effect of varying pH on conjugation efficiency was examined using aseries of phosphate buffers prepared by mixing 0.2M Na₂HPO₄ and 0.2MNaH₂PO₄ in varying proportions: 10:0 (buffer P1, pH 9.29); 9:1 (bufferP2, pH 7.72); 4:1 (buffer P3, pH 7.38); 7:3 (buffer P4, pH 7.14); 3:2(buffer P5, pH 6.94), 1:1 (buffer P6, pH 6.75) 2:3 (buffer P7, pH 6.58);3:7 (buffer P8, pH 6.39); 1:4 (buffer P9, pH 6.12); 1:9 (buffer P10, pH5.81); 0:10 (buffer P11, pH 4.29). Ab1 (prepared as described in Example4) and mal-HRP (2.5 mg/ml) (from Example 1; lyophilisate reconstitutedwith water) were mixed 1:1 and 20 μl aliquots were dispensed intoEppendorf tubes. Each tube then received buffer (one from P1-P11) (20μl) followed by 10 μl of 5 mM TG (i.e. 1 mM final concentration). After60 minutes, 950 μl of TBS/0.1% BSA, pH 8.0 was added and samples wereanalysed by ELISA using rabbit IgG-coated plates (see Example 3). Theresults are shown in FIG. 2.

As can be seen in FIG. 2, active conjugates could be produced under awide variety of conditions and only at extremes of pH were the reactionsrather inefficient. Interestingly, the optimum pH (^(˜)7) issubstantially below the pH typically used for thiolation of biomoleculeswith 2IT. A pH of 8.0 or above is commonly currently used, which tendsto deprotonate amines and increase the rate of reaction with 2IT.However, for in situ thiolation with 2IT in the presence of amaleimide-activated biomolecule, these conditions are clearly notoptimal. This might be explained by increased hydrolysis of maleimidefunctions on mal-HRP at higher pH values and/or increased hydrolysis of2IT. Both of these processes would reduce the efficiency of conjugationreactions between mal-HRP and thiolated Ab1.

Example 6

The effect of varying pH on conjugation efficiency using mal-GOX wasexamined as described in Example 5 except that the samples volumes werehalved and the reactions were terminated by addition of 975 μl ofTBS/0.1% BSA. Samples were analysed by ELISA using rabbit IgG-coatedplates (Example 3). Results are shown in FIG. 3.

As can be seen, the pH optimum for conjugating Ab1 and Gox was similarto that seen for the HRP label (Example 5).

Example 7

The effect of varying buffer species at a fixed pH value of 7.4 wasexamined. Buffer (200 mM) and Ab1 were mixed 1:1 and 10 μl aliquots weredispensed into Eppendorf tubes, followed by 5 μl of 2.5 mg/ml mal-HRP(Example 1) and 5 μl of 5 mM 2IT. Controls reactions were set up with1.2 mM HCl instead of 2IT. The final concentration of each buffer (Tris,HEPES or sodium phosphate) was 50 mM. Results are shown in FIG. 4.

As can be seen in FIG. 4, conjugations in the presence of eitherphosphate or HEPES buffer yielded conjugates that showed similarperformance by ELISA. Despite the 40-fold molar excess of Tris over 2IT,the absorbancy value for the conjugate prepared in the presence of Triswas reduced by only a factor of ^(˜)2. Low absorbance values were seenif 2IT was omitted. In a separate analogous experiment, a conjugateprepared at pH 7.0 in MOPS buffer gave similar ELISA results to aconjugate prepared using sodium phosphate buffer at the same pH (notshown). Thus conjugation reactions may be carried out in several buffersthat lack amine functions, and even in the presence of Tris with amodest loss of performance. This effect of Tris is probably explained byamine-induced ring-opening of 2IT (see Example 8) and competitionbetween the free thiols generated and thiolated Ab1 for mal-HRP.

Example 8

The effect of pH on the release of thiols from 2IT was explored using aseries of phosphate buffers or Tris buffers. 90 μl samples of eachbuffer were mixed with 10 μl of 100 mM 2IT and duplicate aliquots (20μl) were incubated in a clear microplate for 45 min at 25° C. DTNB 200μl (from 80 μg/ml stock in 200 mM sodium phosphate, 1 mM EDTA, pH 8.0)was added and plates were read at A₄₀₅ after 1 minute. Results are shownin FIG. 5.

As can be seen in FIG. 5, with phosphate buffer the release of thiolbecomes more marked as the pH rises, which can be attributed tohydrolysis of 2IT. A pH value of pH 8 or greater is often currently usedfor thiolation of biomolecules with 2IT. At low pH, 2IT is very stable.For any fixed pH value, the rate of thiol production is greater in thepresence of Tris compared with that in the presence of phosphate, whichis consistent with the results in Example 7, which showed reducedefficiency of conjugation in the presence of Tris.

Example 9

The rate of conjugate formation in phosphate buffer was examined atthree different pH values. 50 μl reactions comprised 10 μl of Ab1(Example 4), 20 μl of buffer, 10 μl of mal-HRP (Example 1) (2.5. mg/ml)and 10 μl of 1 mM 2IT. At specified time points (5 min, 20 min, 60 minand 2 h) 5 μl samples were withdrawn and diluted 1/200 in TBS/0.1% BSAprior to testing by ELISA with a rabbit IgG coated plate (Example 3).Results are shown in FIG. 6.

As can be seen in FIG. 6, the rate of conjugate production is pHdependent. A steady increase in absorbancy with time is observed for thepH 6.39 and pH 7.14 incubations over the first four hours and two hours,respectively. The initial rate of increase in absorbancy is greatest atpH 8.15 but the rate slows after 20 minutes and absorbancy value for thepH 7.14 incubation overtakes that of the pH 8.15 incubation after 1hour. Ultimately, the absorbancy value for the low pH incubation alsoexceeded that for the pH 8.15 incubation (data not shown).

Example 10

HRP (2.5 mg/ml) and buffer (P4; Example 5) (samples 1 through 4) or Ab1(Example 4) and P4 buffer (samples 9 through 6) were mixed (1:1) and 10μl portions were mixed with 5 μl of 2IT (1 mM) by staggered addition of2IT at −30 min, −15 min, −5 min and −1 min, relative to time=0 min, atwhich point any outstanding materials (either Ab1 or mal-HRP) wereadded. This allowed in one half of the experiment the formation ofthiolated Ab1 prior to the addition of mal-HRP, and in the other halfpotential polymerisation of mal-HRP prior to the introduction of Ab1. Areference sample (tube 5) was generated with concurrent addition of Ab1and HRP to 2IT at time=0 min. After a further 60 min of incubation, 975μl of TBS/0.1% BSA was added and samples were tested by ELISA using arabbit IgG coated plate (Example 3). Results are shown in FIG. 7.

As can be seen in FIG. 7, the conjugates while not necessarilyphysically identical, all gave very similar absorbance values by ELISA,which suggests that the order of addition is not critical in thisparticular experiment. Importantly, the ability to combine mal-HRP and2IT for significant periods of time without negative effects suggeststhat the two might easily be combined and lyophilised, in order to allowa simple one-step conjugation procedure in which a solution of themolecule to be labelled is used to reconstitute the lyophilised mixture.

Example 11 Preparation of EDBA-Modified and Ethanolamine-ModifiedEnzymes

160 μl of sodium periodate (0.1M) was added to 2 ml of HRP (12.5 mg/mlin 0.1M Na phosphate pH 7.2) and incubated in the dark for 25 min at 25°C. The resulting aldehyde-HRP was desalted on Sephadex G-25 to removeexcess periodate and reacted with 2,2′ (ethylenedioxy)bis-ethylamine(“EDBA” final concentration by volume of 1%) in 0.5M sodium bicarbonate,pH 9.2. After 1 h at RT, sodium cyanoborohydride was added to 50 mM(from 5M stock) to stabilise Schiff bases. After a further 1 h theEDBA-modified HRP sample was desalted into 0.1M sodium phosphate pH 7.2and adjusted to 5 mg/ml.

Portions of HRP were also modified with ethanolamine (which generatesterminal hydroxyls rather than amines) using essentially the sameprocedure to provide control material, OLA-HRP. EDBA- and OLA-modifiedHRP typically contained 13 and 2 TNBS-reactive amines, respectively(i.e. provided B-Y_(n) molecules in which the value of n was,respectively, 13 and 2). Analogous derivatives of Glucose Oxidase (Gox)were prepared with using the same procedure.

EDBA- and OLA-modified HRP (5 mg/ml) were maleimide-activated using 4 mMsulfo-SMCC (as described in Example 1) to generate mal-EDBA-HRP andmal-OLA-HRP, respectively. Samples were desalted into weak buffer (10 mMsodium phosphate pH 7.2) to facilitate subsequent adjustment of pH byaddition of more concentrated buffer solutions.

In some experiments, analogous activation reactions were carried out onEDBA-HRP using 4 mM iodoacetic acid succinimidyl ester to introduceiodoacetyl rather than maleimide functions into HRP.

Example 12 Comparison of Mal-EDBA-HRP and Mal-OLA-HRP in ConjugationReactions

Mal-EDBA-HRP (prepared from two different batches of EDBA-HRP, andmal-OLA-HRP (from Example 11) (10 μl; 25 μg) were each conjugated with 2μl of 5 mg/ml Goat anti-rabbit IgG (in 200 mM Hepes, pH 7.5) and 1.3 μlof 8 mM TG1. After four hours at 25° C. samples were diluted to 1 mlwith TBS/0.1% BSA, from which serial dilutions were prepared and testedin ELISA using either a rabbit IgG coated plate or a control plate (withno rabbit IgG). The results are shown in FIG. 8.

Titration curves for conjugates derived from mal-EDBA-HRP were verysimilar to one another with OD values in excess of 1.5 at 1/10,000dilution. By contrast the conjugate prepared with the mal-OLA-HRPrequired-10-fold higher concentration to achieve similar absorbancevalues. Data for the control plate (i.e. with no antigen) aresuperimposed on one another and show baseline readings over the fullrange of dilutions tested. Thus the strategy of introducing more aminefunctions prior to maleimide-activation significantly enhances theperformance of conjugates in ELISA.

Example 13 Assessment of pH Optimum for Conjugations with Mal-EDBA-HRP

Mal-EDBA-HRP (10 μl) prepared as described above was mixed with 5 μl of2 mg/ml Goat anti-rabbit IgG (20 mM Na phosphate/150 mM NaCl), 2 ul ofTG1 (8 mM) and 3 μl of one of the following 1M buffers: MOPS pH 6.5,MOPS pH 7, Hepes pH 7.5, or EPPS pH 8). After overnight incubation at25° C., serial dilutions were prepared and tested in ELISA using eithera rabbit IgG coated plate or a control plate (with no rabbit IgG). Theresults are shown in FIG. 9.

A very broad pH optimum was observed in reactions with mal-EDBA-HRP (pH6.5-7.5) with the conjugate prepared at pH 7 being only marginallybetter than those prepared at either pH 6.5 or pH 7.5. Thus, addition ofextra maleimide functions has the effect of making conjugation reactionsmore robust to changes in pH compared with reactions carried out withnon-diamine treated HRP (compare Example 5).

Example 14 Comparison of Mal-EDBA-Gox and Mal-OLA-Gox in ConjugationReactions

The applicability of EDBA-treatment in enhancing performance ofconjugates is further illustrated with glucose oxidase, which naturallyhas more available amines than HRP. Nevertheless, periodate oxidationcoupled with EDBA treatment affords conjugates that are substantiallybetter than control conjugates (treated with ethanolamine) in whichhydroxyls rather than amines are appended.

EDBA-Gox-Ig and OLA-Gox-Ig conjugates were prepared and tested in anELISA, as described in Examples 11 and 12. The results are presented inFIG. 10.

Example 15 Conjugation Reactions with Iodoacetyl-HRP

Iodoacetyl-EDBA-HRP (prepared as described in Example 11) (10 μl; 25 μg)was conjugated with 2 μl of 5 mg/ml Goat anti-rabbit IgG (in 200 mMHepes, pH 7.5) and 1.3 μl of 8 mM TG1. After overnight incubation(^(˜)16 hours) in the dark at 25° C. the conjugate was diluted to 1 mlwith TBS/0.1% BSA, from which serial dilutions were prepared and testedin ELISA using either a rabbit IgG coated plate or a control plate (withno rabbit IgG). The results are shown in FIG. 11.

This illustrates that the methods of the present invention are notlimited to electrophilic addition type reactions as exemplified bymaleimides but also displacement reactions with haloacetyl derivatives.

Example 16 Effect of Varying Ab:Enzyme Ratio

Either mal-EDBA-HRP or mal-OLA-HRP (10 μl; 25 ug) was mixed with 10 μlof Goat and rabbit IgG of varying concentration (in 200 mM Hepes pH 7.5,and 2 ul of TG1 (8 mM). After 4 h incubation at 25° C., samples werediluted to 250 ng of antibody per ml and tested in ELISA using either arabbit IgG coated plate or a control plate (with no rabbit IgG). Theresults are presented in FIG. 12.

As can be seen in FIG. 12, at all antibody:HRP ratios, the conjugatesprepared with mal-EDBA-HRP show much higher absorbance values than thoseprepared with mal-OLA-HRP. The absorbance values are slightly lower withhigh ratios of antibody to HRP, presumably because the number of HRPmolecules attached cannot exceed 2 per molecule of antibody (2:1 weightratio=^(˜)1:2 molar ratio), whereas higher numbers may be attached withlower ratios of antibody to HRP. Thus conjugates of lower molecularweight, which might be advantageous in applications that requirepenetration into tissues (e.g. as in immunohistochemistry) are favouredby higher antibody:HRP ratios.

It is apparent from FIG. 12 that increasing the ratio of mal-OLA-HRP toAb (by reducing the amount of antibody in each reaction) has only amodest effect on conjugate performance. Even with a substantial excessof mal-OLA-HRP (16:1 by weight; ^(˜)64:1 molar ratio) the efficiency ofconjugation never approaches that observed with mal-EDBA-HRP. Since therate of production of thiolated antibody at any fixed Ab-HRP ratio isthe same with both types of HRP some other process must operate to limitconjugation efficiency in the case of excess mal-OLA-HRP. If we considerthe more general case of A reacting with B-Y, TG1 reacts with amines onA to generate A-SH. It also reacts with water in a competing hydrolysisreaction, which gets faster with increasing pH, especially above pH 7,to generate unwanted free thiols.

In FIG. 12, since a large excess mal-OLA-HRP gives conjugates thatclearly show sub optimal performance, the concentration of unwanted freethiols must reach a critical point (i.e. where conjugation efficiency iscompromised) before all of the molecules of Ab-NH₂ have been convertedinto Ab-SH. In the example, the concentration of thiol generator (TG) is800 μM. The concentration of the amine reactant is ^(˜)6 μM (i.e. for 1mg/ml antibody), or effectively 60 μM amine, (assuming about 10 lysinesare capable of reacting with TG). In the case of mal-OLA-HRP theconcentration of maleimide functions cannot be any greater than theinitial amine content prior to SMCC treatment and is therefore nogreater than ^(˜)50-100 μM. Although amines are more nucleophilic thanwater molecules at physiological pH values, the concentrations ofreactants and the solvent (i.e. water) are unfavourable in typicalconjugation reactions.

Thus merely increasing the ratio of HRP to Ab is not enough to overcomeundesirable competing reactions. There have to be sufficient maleimidesto cope with a more rapid release of free thiol (i.e. TG hydrolysis)than A-SH production in the conjugation reaction. Although amines on Aare more reactive than the hydroxyl groups of water molecules, a veryhigh concentration of water (55 M) is available to attack TG. Thisunderlines why multivalent HRP is especially effective as it can reactwith unwanted thiols that are generated throughout the 2-3 hours'conjugation reaction and yet still react with A-SH.

Example 17

To investigate various amines as potential quench agents that might beused, if required, to halt conjugation reactions, we examined thiolrelease from 2IT using a standard DTNB assay. 5 mM solutions of glycine,ethanolamine, or 1,3-diaminopropane (DAP) were prepared in 100 mM sodiumphosphate buffer, pH 7.4. 980 μl aliquots of each buffer were mixed with20 μl of 100 mM 2IT in 1.2 mM HCl. Freshly prepared DTNB reagent (200 μlof 80 μg/ml solution in 10 mM sodium phosphate, 1 mM EDTA, pH 8.0) wasadded to 20 μl aliquots of each reaction mix over a time course. Sampleswere read within 1 minute of DTNB addition. Results are shown in FIG.13.

The amines that were selected, ethanolamine, glycine and DAP,additionally contain neutral, acidic, and basic groups, respectively. Itis important to note that the data in FIG. 14 potentially represent thenet effect of two opposing pathways (i) thiol release from 2IT (ii)intramolecular reaction of released thiol. This is particularly evidentwith glycine where the amount of thiol begins to fall after one hour.The highest ‘apparent’ rate is with DAP, but despite its extra aminefunction compared with glycine or ethanolamine this appears not toexplain greater thiol release (see Example 18) but rather a slowerintramolecular reaction, which is perhaps connected with the extrapositive charge that is introduced into the product of the initialring-opening reaction.

Example 18

To get a better appreciation of the initial rates of thiol release withpotential quenchers, the experiment of Example 17 was modified to allowimmediate ‘capture’ of any released thiol by reaction with DTNB. Becauseof the need to measure DTNB reactions at pH 8, it was not possible touse the same pH as in Example 17, but the same three amines werestudied. DTNB (80 μg/ml) was freshly prepared in 0.2M sodium phosphate,1 mM EDTA, pH 8.0, and dispensed (200 μl per well) into a clear 96-wellplate. 20 μl of 100 mM 2IT was added and the absorbancy was read at A₄₀₅every 2 min using an automatic plate cycling function. Results are shownin FIG. 14.

As can be seen in FIG. 14, the three amines induce the release of thiolsat very similar rates, though glycine is the fastest. Since glycine alsoappears to support a relatively fast intramolecular reaction that leadsto disappearance of thiols (Example 17), it is the most promising of thethree amines for halting conjugation reactions. A significant release ofthiols under conditions normally used for thiolation of biomolecules(i.e. the control reaction in this experiment) is also evident.

Example 19

Freeze-drying is commonly employed to stabilize and extend the shelflife of protein-based products. Excipients that are often used to helpto stabilize the active constituents include cryoprotectants, whoseprimary function is to afford protection during the freezing step, andlyoprotectants, whose function is to prevent degradation during freezedrying and/or during storage. For any new sample, the best formulationfor freeze-drying can only be determined empirically. There are noprecedents, as far as we are aware, for freeze-drying of TG or forfreeze-drying of TG in the presence of B-Y molecules. Freeze-drying ofmixtures of TG and B-Y requires the development of a single formulationto stabilize Y functions (e.g. maleimides), TG (e.g. 2-IT) and thebiological activity of B (e.g. HRP, alkaline phosphatase). Clearly, ifthere is significant damage to one active ingredient any subsequentconjugation reactions will be compromised even if the other componentsare well preserved.

In initial experiments with a range of buffers of varying compositionand pH, the extent of unwanted ring opening of 2-IT (alone) duringfreeze drying was assessed by measuring free thiol content of thefreeze-dried material using DTNB reagent. The release of free thiolsappeared to be dependent on the pH of the solution prior tofreeze-drying. Acidic solutions, including dilute hydrochloric acid (1.2mM-12 mM), 20 mM sodium phosphate (pH<6.5) and 20 mM sodium acetate (pH5) gave freeze-dried materials with low free thiol content, consistentwith the previously noted greater solution stability of 2-IT at low pH(Example 8). Buffers commonly used for conjugation reactions (i.e. thosebased on neutral or slightly basic solutions of phosphate, NaCl andEDTA) were relatively poor stabilizers of 2-IT in freeze-drying, as wereother buffers of pH>6.5.

An unexpected observation was made when the freeze-dried 2-IT sampleswere left incubating with DTNB reagent for several hours. Normally,because 2-IT is unstable at the pH used for the DTNB reaction (pH 8),there is a time-dependent release of thiols, and thus a risingbackground signal. Despite the apparent stabilization of 2-IT in sodiumacetate (i.e. absence of thiols after free-drying), the expectedtime-dependent increase in the background signal was largely absent.This observation was not explained by any direct inhibitory effect ofacetate on the reaction of DTNB with thiols. The presumed chemicaltransformation of TG1 that prevented the release of thiol groups was notfurther investigated, but it may be similar to the secondary reactionobserved under other conditions for TG1 (see p 4), which leads to theformation of a unreactive thioether. Conjugates prepared withmal-HRP/TG1 mixtures that were freeze dried in the presence of 4 mMsodium phosphate, pH 5.8, gave far stronger signals in ELISA than thosefreeze dried in the presence of 5 mM sodium acetate pH 5. Taken togetherthe results illustrate that while low pH is necessary for stabilizationof TG1 during freeze-drying (and thus for efficient conjugate formationsubsequently carried out at slightly alkaline pH), a low pH alone is notsufficient to preserve the integrity of TG1.

In a preferred embodiment of the present invention the buffer forfreeze-drying mixtures of B-Y/TG1 comprises sodium phosphate. The pH ofthe buffer is preferably below 6.5 and more preferably below 6.0.

As it is necessary to raise the pH of the freeze-dried mixture uponreconstitution with a buffered solution of A in order to provide optimalconditions for bioconjugation reactions, weakly buffered freeze-driedmixtures are preferred. Preferably the buffer concentration is below 200mM; more preferably below 50 mM and most preferably below 20 mM.

In a preferred embodiment of the present invention, the mixture offreeze-dried TG/B-Y/phosphate buffer also contains a polyol, such as asugar or dextran, or combinations of polyols. Most preferably the polyolis trehalose. The concentration of trehalose is preferably >1%, and mostpreferably around 5% (w/v).

In a particularly preferred embodiment, where B-Y is either HRP oralkaline phosphatase, the mixture also contains a metal ion or metalions. In a particularly preferred embodiment the metal ion is Ca²⁺ orMg²⁺ (typically added as MgCl₂), preferably in the range 1-10 mM Mg²⁺,desirably about 5 mM.

Example 19.1 Freeze Drying of Maleimide-Activated Enzymes with 2-IT

Alkaline phosphatase (16.2 mg/ml; Biozyme code ALP112G) in 5 mM Tris/5mM MgCl₂/0.1 mM ZnCl₂, pH 7.0 was diluted to 5 mg/ml with 0.1M sodiumphosphate pH 7.2 and activated with 4 mM sulfo-SMCC for 1 h at 25° C.The sample was then desalted into 10 mM sodium phosphate pH 5.8 to givea concentration of 3.125 mg/ml. 2. Excipients were added, as required,and 2-IT was added last typically at 400 or 800 μM concentration. Thefinal concentration of enzyme prior to freeze drying was 2.5 mg/ml.Samples were rapidly frozen in liquid nitrogen in polypropylene tubes orglass vials prior to freeze drying in an Advantage ES freeze dryer usinga 24 hour cycle: Step 1, shelf temperature −40° C. for 1320 minutes,step 2 shelf temperature −10° C. for 60 minutes and step 3 shelftemperature +20° C., 60 minutes. Following freeze-drying, samples werestored at either −20 or 37° C. for several days.

EDBA-modified HRP was prepared as in Example 11, and activated with SMCCas described above. The sample was desalted as described above into 10mM sodium phosphate pH 5.8 and further prepared for freeze-drying asdescribed for alkaline phosphatase.

Example 20 Stability of Freeze Dried Mal-HRP/2-IT Mixtures

Maleimide-activated EDBA-modified HRP was desalted into pH 5.8 buffer(as described above, Example 19) and TG1 was added to give a finalconcentration of 800 μM prior to freeze-drying of 40 μl portions (100 μgHRP). 320 μl of 1 mg/ml Goat anti-mouse IgG antibody in 20 mM sodiumphosphate/150 mM NaCl, pH 7.2, was supplemented with 32 μl of 2MHepes/10 mM EDTA, pH 7.25. 100 μl of this material was used toreconstitute each vial of the freeze-dried mixture. Conjugation wasallowed to proceed at 25° C. overnight and the resulting conjugates weretested by ELISA on a mouse IgG-coated plate prepared as described inExample 3.

The results are illustrated in FIG. 15, which is a graph of absorbancyat 405 nm (in arbitrary units) against a log scale of conjugatedilution. The graph shows the titration of conjugates prepared asdescribed in this example using lyophilised mixtures which wereincubated overnight, prior to conjugation, at −20° C. (solid circles),25° C. (open circles) or 37° C. (solid squares).

As can be seen, as the lyophilized mixture is subjected to increasingtemperature there is a marked loss of performance of the resultingconjugates. At 1/10,000 dilution the absorbance signal for conjugateprepared with mixture stored overnight at 37° C. is ^(˜)20% of that forthe conjugate prepared with mixture stored at −20° C., and the dilutionof conjugate required to give an absorbance of 1.0 is shifted by anorder of magnitude.

Example 21 Stabilization of Freeze Dried Mal-Enzyme/2-IT

Sugars often help to stabilize proteins during freeze drying and/orstorage, and the inclusion of trehalose in freeze-dried mixtures ofmaleimide-EDBA-HRP/TG and maleimide-alkaline phosphatase/TG at 37° C.significantly increased stability compared with samples lacking thesugar (as measured by performance in ELISA of conjugates prepared fromthe mixtures) (see Table below and Example 20). Since alkalinephosphatase is commonly assayed in the presence of Mg²⁺ and Zn²⁺ ions weconsidered the possibility that metal ions might further help tostabilize this enzyme during freeze-drying or storage. However, sincemetal ions might have damaging effects on other components (e.g. 2-IT)during freeze-drying, or might interfere with subsequent conjugationreactions, we performed some initial trials using HRP, which is lessexpensive than alkaline phosphatase.

Freeze dried mixtures were prepared as described in Example 19containing malemide-activated EDBA-modified HRP in 10 mM sodiumphosphate buffer, pH 5.8, plus trehalose 5% (w/v), metal ions (as notedbelow), and 2-IT (400 μM). Mixtures were stored at 37° C. prior toconjugation. To our surprise, for HRP samples that were spiked with 5 mMMgCl₂ (which is not required for activity of HRP) and then freeze-dried,the stability at 37° C. of the freeze-dried mixture markedly improved,as judged by performance in ELISA of Goat anti-rabbit IgG-HRP conjugatesprepared with the mixture, and tested as described in Example 3. Insubsequent studies with trehalose/2-IT/maleimide-alkaline phosphatasemixtures, we found that MgCl₂ had not only a protective action onalkaline phosphatase during storage at elevated temperature but also acryoprotective action. These results and those for other metal ions aresummarized in the table below.

Stabilization of Mal-EDBA-HRP/2-IT and Mal-Alkaline Phosphatase/2-ITMixtures at 37° C. as Measured by Conjugate Performance in ELISA

% Enzyme Additive Trehalose (−20° C.) Mal-EDBA-HRP Trehalose 25Mal-EDBA-HRP Trehalose/Mg²⁺ (5 mM) 77 Mal-EDBA-HRP Trehalose/Zn²⁺ (0.5mM) 34 Mal-EDBA-HRP Trehalose/Ca²⁺ (5 mM) 84 Mal-alkaline phosphataseTrehalose 37 Mal-alkaline phosphatase Trehalose/Mg²⁺ (5 mM) 104 Mal-alkaline phosphatase Trehalose/Zn²⁺ (0.5 mM) 44 Mal-alkalinephosphatase Trehalose/Mg²⁺ (5 mM)/ 102  Zn²⁺ (0.5 mM) Mal-alkalinephosphatase Trehalose/Mg²⁺ (5 mM) 148* *Mg²⁺/trehalose at −20° C. versustrehalose at −20° C.

Freeze-dried mixtures were incubated at 37° C. for 6 days (HRP) or 5days (alkaline phosphatase) and then used to prepare goat anti-rabbitconjugates that were tested in a rabbit IgG ELISA. The % activity valueswere determined by dividing the ELISA absorbance value for a 1/10,000dilution of conjugate prepared from material stored at 37° C. by theabsorbance value obtained for a trehalose formulation (i.e. with nometal ions) prepared from material stored at −20° C., unless otherwisestated.

As can be seen, trehalose improves the stability of the mixture toelevated temperature (the 75% loss of activity over 6 days [1^(st) lineof the table] is broadly similar to that over 1 day without trehalose;see Example 20). Mg²⁺ further significantly improves the stability ofmal-EDBA-HRP/2-IT/trehalose mixtures, and there is only a 23% loss ofactivity following incubation for 6 days at 37° C. of the freeze-driedmixture that was used to prepare conjugate. In respect of a conjugationkit comprising freeze-dried materials, the formulation with trehaloseand Mg²⁺ greatly facilitates transportation at ambient temperatures.Calcium (5 mM) also stabilized mal-EDBA-HRP/2-IT/trehalose mixtures, butZn²⁺ at concentrations commonly employed in assays of alkalinephosphatase (0.5 mM) had little protective effect.

A similar pattern emerges with alkaline phosphatase, with a markedimprovement in the ELISA reactivity of resulting conjugates if themal-alkaline phosphatase/2-IT mixture is freeze dried in presence oftrehalose and Mg²⁺. Compared with formulations containing trehalosealone and stored at −20° C., there is apparently no loss of activityafter 5 days of storage at 37° C. Unlike HRP, however, where thebeneficial effect of the excipients is seen at elevated temperature,rather than during freeze-drying, mal-alkaline phosphatase/2-IT mixturesare also protected by the excipients during freezing/freeze-drying.Thus, samples freeze-dried in the presence of Mg²⁺ are substantiallymore active immediately after freeze drying than those in the absence ofthe metal ion.

The invention claimed is:
 1. A method of reacting a polypeptide firstchemical entity and an enzyme or fluorescent material second chemicalentity to form a conjugate in which the first and second chemicalentities are covalently bound with respect to each other, comprising thesteps of forming a conjugation reaction mixture by bringing intosimultaneous contact in aqueous conditions, the first chemical entity,the second chemical entity and a thiol generator, wherein the thiolgenerator reacts with the polypeptide first chemical entity in athiolation reaction resulting in formation of a free sulfhydryl group onthe polypeptide first chemical entity, wherein reaction of the thiolgenerator with a primary amine on the polypeptide first chemical entitytakes place at a pH below 8, and the free sulfhydryl group reacts withthe enzyme or fluorescent second chemical entity to form the conjugate,and wherein the enzyme or fluorescent second chemical entity is modifiedby the attachment of at least two molecules that bind sulfhydryl groupsand is therefore polyvalent with respect to its reactivity withsulfhydryl groups, and further wherein the molar ratio of the enzyme orfluorescent second chemical entity to thiol generator is 1:1 or less. 2.The method according to claim 1, wherein the thiol generator comprisesone or more of a thiolactone, an iminothiolactone, an episulfide and athiazolidine.
 3. The method according to claim 2, wherein the thiolgenerator comprises 2-iminothiolane.
 4. The method according to claim 1,wherein the polypeptide first chemical entity, the enzyme or fluorescentmaterial second chemical entity and the thiol generator are combinedsimultaneously in a one step procedure.
 5. The method according to claim1, comprising a first step in which the thiol generator and thepolypeptide first chemical entity are combined, and a second step inwhich the enzyme or fluorescent material second chemical entity iscombined with the thiol generator and first chemical entity.
 6. Themethod according to claim 1, comprising a first step in which thepolypeptide first and enzyme or fluorescent material second chemicalentities are combined, and a second step in which the thiol generator iscombined with the polypeptide first and enzyme or fluorescent materialsecond chemical entities.
 7. The method according to claim 1, comprisinga first step in which the thiol generator and enzyme or fluorescentmaterial second chemical entity are combined, and a second step in whichthe polypeptide first chemical entity is combined with the thiolgenerator and the enzyme or fluorescent material second chemical entity.8. The method according to claim 7, wherein the polypeptide firstchemical entity is added in liquid form to a dried mixture comprisingthe enzyme or fluorescent material second chemical entity and the thiolgenerator.
 9. The method according to claim 1, wherein the thiolgenerator has a free thiol group content of less than 5% in molar terms.10. The method according to claim 1, wherein the thiol generator ispresent in up to 120 times molar excess in relation to the polypeptidefirst chemical entity.
 11. The method according to claim 1, wherein theenzyme or fluorescent material second chemical entity is present in upto 10 times molar excess in relation to the polypeptide first chemicalentity.
 12. The method according to claim 1, wherein the enzyme orfluorescent material second chemical entity comprises a polymer.
 13. Themethod according to claim 1, wherein the enzyme or fluorescent materialsecond chemical entity comprises a polypeptide.
 14. The method accordingto claim 1, wherein the enzyme or fluorescent material second chemicalentity comprises an enzyme.
 15. The method according to claim 1, whereinthe enzyme or fluorescent material second chemical entity comprises amolecule selected from the group consisting of: horseradish peroxidase,alkaline phosphatase and glucose oxidase.
 16. The method according toclaim 1, wherein the enzyme or fluorescent material second chemicalentity comprises five or more sulfhydryl-reactive groups per molecule.17. The method according to claim 1, wherein the enzyme or fluorescentmaterial second chemical entity comprises ten or moresulfhydryl-reactive groups per molecule.
 18. The method according toclaim 1, wherein the enzyme or fluorescent material second chemicalentity comprises from five to fifteen sulfhydryl-reactive groups permolecule.
 19. The method according to claim 1, wherein the molar ratioof second chemical entity to thiol generator is less than 1:1.
 20. Themethod according to claim 19, wherein the molar ratio is of secondchemical entity to thiol generator in the range 1:1 to 1:20.
 21. Themethod according to claim 19, wherein the molar ratio of second chemicalentity to thiol generator is in the range 1:10 to 1:15.
 22. The methodaccording to claim 1, wherein reaction of the thiol generator with aprimary amine on the polypeptide first chemical entity takes place at apH below 7.8.
 23. The method according to claim 1, wherein the reactionof the thiol generator with a primary amine on the polypeptide firstchemical entity takes place at a pH below 7.7.
 24. The method accordingto claim 9, wherein the thiol generator has a free thiol group contentof less than 3% in molar terms.
 25. The method according to claim 9,wherein the thiol generator has a free thiol group content of less than1% in molar terms.
 26. The method according to claim 1, furthercomprising adding a nucleophile to terminate the conjugation reaction.27. A method of reacting a polypeptide first chemical entity and anenzyme or fluorescent material second chemical entity to form aconjugate in which the first and second chemical entities are covalentlybound with respect to each other, comprising the steps of forming aconjugation reaction mixture by bringing into simultaneous contact inaqueous conditions, the first chemical entity, the second chemicalentity and a thiol generator, wherein the thiol generator reacts withthe polypeptide first chemical entity in a thiolation reaction resultingin formation of a free sulfhydryl group on the polypeptide firstchemical entity, and the free sulfhydryl group reacts with the enzyme orfluorescent second chemical entity to form the conjugate, and whereinthe enzyme or fluorescent second chemical entity is polyvalent withrespect to its reactivity with sulfhydryl groups, and further whereinthe molar ratio of the enzyme or fluorescent second chemical entity tothiol generator is 1:1 or less; and terminating the formation of theconjugate by adding a nucleophile to the conjugation reaction mixture.